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No âmbito dos Estudos da Tradução Audiovisual, vários estudos têm sido dedicados à investigação da tradução da linguagem tabu, na área da dobragem, da legendagem, o fansubbing, e mais recentemente até na área da Acessibilidade. Contudo, esta investigação tem se visto restrita à análise de textos, sendo mais escassa à investigação do agente humano no processo da tradução. Neste enquadramento, este estudo empírico tem por objetivo investigar as atitudes de tradutores audiovisuais relativamente à tradução para legendagem na televisão de sinal aberto em Portugal. Aplicou-se, com esse fim, um questionário a tradutores. Os resultados demostraram um alto nível de concordância com a manutenção do tabu, tal como a relevância de diferentes aspectos relacionados com o papel que o tabu assume nas narrativas fílmicas. Não obstante, estas atitudes diferem da estratégia mais comum na tradução e legendagem de tabu no contexto português, a omissão, o que pode indiciar um novo paradigma na tradução de linguagem tabu.

Audiovisual media reflect language use in the community and the context of attitudes and stereotypes regarding different language varieties. Against this backdrop, taboo language has become a frequent resource for linguistic characterisation in cinema. Studies related to taboo language in audiovisual contexts suggest some functions of these words in films, though not systematically nor layered. Based on the work of Allan and Burridge (“Swearing”) on the functions of taboo language in its authentic use and Delabastita (“Great Feast of Languages”) on the extratextual functions of multilingualism in Shakespearean work, this article offers an empirical, multidisciplinary, systematic approach to the use of taboo language in films. We propose a typology of four intratextual and three extratextual functions of taboo language in audiovisual contexts. This typology will then be tested on a corpus of films via a detailed multimodal quantitative and qualitative analysis.

Introdução: As atribuições do farmacêutico na execução de atividades de ensaios clínicos envolvem o processo de gerenciamentos dos produtos sob investigação. Dessa forma, caracterizam-se as responsabilidades pelo recebimento, armazenamento e controle desses produtos e por todo o suporte ao delineamento da pesquisa, junto aos demais profissionais da equipe designada para o estudo. Adicionalmente, estão inseridos as atividades de dispensação dos produtos sob investigação e o preparo, bem como acompanhamento da adesão terapêutica do participante, possíveis reações adversas ao tratamento proposto, orientações sobre o tratamento e realização das comunicações periódicas das atividades farmacêuticas executadas com os monitores, patrocinadores e investigadores dos estudos em curso. A atuação do farmacêutico nas atividades de ensaios clínicos é de extrema importância para uma condução dos protocolos de pesquisa e contribui para minimizar eventos adversos e desvios de delineamento dos estudos. O impacto da atuação dos farmacêuticos é ainda pouco conhecido. Objetivo: Caracterizar as atividades desenvolvidas pelo farmacêutico em um hospital universitário no primeiro semestre de 2022. Metodologia: Estudo descritivo, retrospectivo, de levantamento histórico das atividades desenvolvidas pela equipe de farmacêuticos do serviço de ensaios clínicos do Setor de Farmácia Hospitalar de um hospital universitário em Salvador-Bahia no primeiro semestre de 2022. Os dados foram contabilizados e aplicada estatística descritiva. Resultados e Discussão. No período avaliado houve doze estudos ativos no centro de pesquisa, sendo que seis tiveram atuação dos farmacêuticos, inseridos nas especialidades de infectologia, imunologia, cardiologia e gastro-hepatologia. Esses protocolos envolveram o atendimento de 120 participantes, com 123 dispensações ambulatoriais de medicamentos. Foram realizadas 06 solicitações e 08 recebimentos de medicamentos no período avaliado. Foram identificadas 02 devoluções de medicamentos para patrocinador e 04 destruições de medicamentos no próprio centro. A comunicação efetiva com patrocinadores para alinhamento e orientações sobre os protocolos clínicos ocorreu em 09 monitorias remotas e 07 monitorias presenciais. Conclusão: A pesquisa clínica é uma atividade multiprofissional e transdisciplinar, que envolve conhecimentos e habilidades específicas dos profissionais, além do trabalho compartilhado em equipe. O farmacêutico é um profissional indispensável na gestão racional dos produtos sob investigação nos ensaios clínicos e suas atividades contribuem para a adequada segurança na condução de estudos em pesquisa clínica.

The accurate quantification of DNA in forensic samples is of utmost importance. These samples are often present in limited amounts; therefore, it is indicated to use the appropriate analysis route with the optimum DNA amount (when possible). Also, DNA quantification can inform about the degradation stage and therefore support the decision on which downstream genotyping method to use. Consequently, DNA quantification aids in getting the best possible results from a forensic sample, considering both its DNA quantity and quality limitations. Here, we introduce NuMY, a new quantitative real-time PCR (qPCR) method for the parallel quantification of human nuclear (n) and mitochondrial (mt) DNA, assessing the male portion in mixtures of both sexes and testing for possible PCR inhibition. NuMY is based on previous work and follows the MIQE guidelines whenever applicable. Although quantification of nuclear (n)DNA by simultaneously analyzing autosomal and male-specific targets is available in commercial qPCR kits, tools that include the quantification of mtDNA are sparse. The quantification of mtDNA has proven relevant for samples with low nDNA content when conventional DNA fingerprinting techniques cannot be followed. Furthermore, the development and use of new massively parallel sequencing assays that combine multiple marker types, i.e., autosomal, Y-chromosomal, and mtDNA, can be optimized when precisely knowing the amount of each DNA component present in the input sample. For high-quality DNA extracts, NuMY provided nDNA results comparable to those of another quantification technique and has also proven to be a reliable tool for challenging, forensically relevant samples such as mixtures, inhibited, and naturally degraded samples.

Abstract Background: The treatment of advanced periocular basal cell carcinomas becomes a challenge as surgery may involve highly mutilating procedures. Vismodegib is the first selective hedgehog inhibitor approved for the treatment of locally advanced tumors or metastatic disease. Objective: Analyze the results of treatment with vismodegib for advanced periocular basal cell carcinomas in a real-life setting of a reference center between 2014 and 2020. Methods: Retrospective longitudinal study. The patient’s demographic profile, comorbidities, tumor characteristics, and treatment outcomes were analyzed. Results: A total of 13 patients were included. Median follow-up and treatment duration were 15.9 and 10.5 months, respectively. Objective clinical response rate was 76.9%: 30.8% had a complete response and 46.2% a partial response. The median duration of response was 13 months. Progressive disease was observed in 38.5% of cases, with a median of 19 months after the beginning of treatment. Eighty-four percent of the patients had at least one adverse event, and 61.54% needed to interrupt treatment temporarily or permanently to increase tolerability. Study limitations: Being a retrospective study in a real-life setting, the evaluation of objective clinical response was subjective to physician appreciation. Conclusion: Vismodegib is a safe and effective treatment for locally advanced basal cell carcinoma. To prevent recurrences, the drug should be used continually when tolerated. The role of neoadjuvant vismodegib before surgery is being investigated and might add an important step in searching for a definitive treatment for these cases.

Introdução: A gestão de materiais e suprimentos possui papel estratégico no contexto da modernização da Farmácia Hospitalar. Diante disso, a gestão da aquisição de medicamentos se afigura como uma importante área de investigação, uma vez que instituições públicas destinam grande volume de recursos financeiros à aquisição desses produtos. Método: O presente estudo teve por fim avaliar o desempenho de fornecedores de medicamentos do Hospital Universitário Dr. Washington Antônio de Barros (HU/UNIVASF), localizado na cidade de Petrolina-PE, segundo os critérios de aceitação estabelecidos no edital do Pregão 30/2015. Trata-se de um estudo transversal de caráter descritivo e prospectivo, realizado entre os meses de dezembro de 2015 a março de 2016. O instrumento utilizado à coleta dos dados foi o “Formulário-Avaliação e Qualificação de Fornecedores”, com avaliação dicotômica, sim ou não, utilizado pelo Setor de Farmácia do HU/UNIVASF. O instrumento possui nove itens: nota fiscal, quantidade de medicamentos entregues, marca, especificação, embalagem, rotulagem, prazo de entrega, validade e horário de entrega. Os dados foram analisados por meio do programa EXCELL-2013. Resultados E Discussão: Entre os 16 fornecedores homologados no Pregão 30/2015, apenas 14 foram avaliados no período do estudo: 21% (03) Indústria Farmacêutica (IF), 79% (11) Distribuidoras de Medicamentos (DM). Observou-se que nenhum fornecedor cumpriu os nove itens, entretanto IF apresentou o melhor desempenho com 77,77% de critérios atendidos. Com relação ao item Prazo de Entrega, 5/11 empresas do grupo DM cumpriram com os critérios estabelecidos, enquanto no grupo IF, apenas 1/3 empresas conseguiram atender as exigências do Edital. No item Validade foi observado o cumprimento dos critérios por 3/11 do grupo DM e 2/3 do grupo IF. Por conseguinte, a análise dos dados demonstrou que ambos fornecedores obtiveram aproveitamento de 100% nos itens Nota Fiscal, Marca, Especificação, Embalagem e Rotulagem. Conclusão: Os resultados do presente estudo demonstraram que existiram, junto ao pregão 30/2015, descumprimentos no período de dezembro de 2015 a março de 2016 pelas empresas fornecedoras de medicamentos. Maior evidência foi observada no item Prazo de Entrega, o que é crucial para o serviço de farmácia, visto que gera o desabastecimento de medicamentos no hospital. Na comparação, entre os tipos de fornecedores as IFs se mostraram melhor qualificadas. Espera-se que os resultados da presente investigação possam estimular e fundamentar outros profissionais do HU-UNIVASF à realização de estudos futuros nas áreas da farmácia hospitalar e gestão de compras.

RESUMO Os falantes interagem através dos recursos linguísticos ao seu dispor para produzir significados ao nível da compreensão do mundo que os rodeia e dos restantes falantes (EGGINS, [1994] 2004, p. 11). O falante conhece, então, um código para cada situação comunicativa e adequa as suas seleções estilísticas ao contexto em que as produz. Neste enquadramento, este artigo exploratório investiga a linguagem tabu do ponto de vista das variáveis do contexto situacional, como propostas por Halliday (1978, [1985] 2014). Segue, por um lado, uma abordagem teórica onde propõe o cruzamento das variáveis do registro com os conceitos de uso marcado e não marcado (HYMES, 1974); e, por outro, aplica esta proposta num corpus escolhido para o efeito. Os resultados, de ordem quantitativa, demonstram o predomínio do uso não marcado da linguagem tabu no corpus.

The high number of matching haplotypes of the most common mitochondrial (mt)DNA lineages are considered to be the greatest limitation for forensic applications. This study investigates the potential to solve this constraint by massively parallel sequencing a large number of mitogenomes that share the most common West Eurasian mtDNA control region (CR) haplotype motif (263G 315.1C 16519C). We augmented a pilot study on 29 to a total of 216 Italian mitogenomes that represents the largest set of the most common CR haplotype compiled from a single country. The extended population sample confirmed and extended the huge coding region diversity behind the most common CR motif. Complete mitogenome sequencing allowed for the detection of 163 distinct haplotypes, raising the power of discrimination from 0 (CR) to 99.6% (mitogenome). The mtDNAs were clustered into 61 named clades of haplogroup H and did not reveal phylogeographic trends within Italy. Rapid individualization approaches for investigative purposes are limited to the most frequent H clades of the dataset, viz. H1, H3, and H7.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications.One Sentence SummaryNanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

The development of microhaplotype (MH) panels for massively parallel sequencing (MPS) platforms is gaining increasing relevance for forensic analysis. Here, we expand the applicability of a 102 autosomal and 11 X-chromosome panel of MHs, previously validated with both MiSeq and Ion S5 MPS platforms and designed for identification purposes. We have broadened reference population data for identification purposes, including data from 240 HGDP-CEPH individuals of native populations from North Africa, the Middle East, Oceania and America. Using the enhanced population data, the panel was evaluated as a marker set for bio-geographical ancestry (BGA) inference, providing a clear differentiation of the five main continental groups of Africa, Europe, East Asia, Native America, and Oceania. An informative degree of differentiation was also achieved for the population variation encompassing North Africa, Middle East, Europe, South Asia, and East Asia. In addition, we explored the potential for individual BGA inference from simple mixed DNA, by simulation of mixed profiles followed by deconvolution of mixture components.

Immigrants from diverse origins have arrived in Paraguay and produced important demographic changes in a territory initially inhabited by indigenous Guarani. Few studies have been performed to estimate the proportion of Native ancestry that is still preserved in Paraguay and the role of females and males in admixture processes. Therefore, 548 individuals from eastern Paraguay were genotyped for three marker sets: mtDNA, Y-SNPs and autosomal AIM-InDels. A genetic homogeneity was found between departments for each set of markers, supported by the demographic data collected, which showed that only 43% of the individuals have the same birthplace as their parents. The results show a sex-biased intermarriage, with higher maternal than paternal Native American ancestry. Within the native mtDNA lineages in Paraguay (87.2% of the total), most haplogroups have a broad distribution across the subcontinent, and only few are concentrated around the Paraná River basin. The frequency distribution of the European paternal lineages in Paraguay (92.2% of the total) showed a major contribution from the Iberian region. In addition to the remaining legacy of the colonial period, the joint analysis of the different types of markers included in this study revealed the impact of post-war migrations on the current genetic background of Paraguay.

Abstract Given the increasing exploration of fluorescent tracers in the field of nuclear medicine, a need has risen for practical development guidelines that can help improve the translation aspects of fluorescent tracers. This editorial discusses the does and don’ts in developing fluorescence tracers. It has been put forward by the European Association of Nuclear Medicine (EANM) Translational Molecular Imaging & Therapy committee and has been approved by the EANM board.

We detail the development of the ancestry informative single nucleotide polymorphisms (SNPs) panel forming part of the VISAGE Basic Tool (BT), which combines 41 appearance predictive SNPs and 112 ancestry predictive SNPs (three SNPs shared between sets) in one massively parallel sequencing (MPS) multiplex, whereas blood-based age analysis using methylation markers is run in a parallel MPS analysis pipeline. The selection of SNPs for the BT ancestry panel focused on established forensic markers that already have a proven track record of good sequencing performance in MPS, and the overall SNP multiplex scale closely matched that of existing forensic MPS assays. SNPs were chosen to differentiate individuals from the five main continental population groups of Africa, Europe, East Asia, America, and Oceania, extended to include differentiation of individuals from South Asia. From analysis of 1000 Genomes and HGDP-CEPH samples from these six population groups, the BT ancestry panel was shown to have no classification error using the Bayes likelihood calculators of the Snipper online analysis portal. The differentiation power of the component ancestry SNPs of BT was balanced as far as possible to avoid bias in the estimation of co-ancestry proportions in individuals with admixed backgrounds. The balancing process led to very similar cumulative population-specific divergence values for Africa, Europe, America, and Oceania, with East Asia being slightly below average, and South Asia an outlier from the other groups. Comparisons were made of the African, European, and Native American estimated co-ancestry proportions in the six admixed 1000 Genomes populations, using the BT ancestry panel SNPs and 572,000 Affymetrix Human Origins array SNPs. Very similar co-ancestry proportions were observed down to a minimum value of 10%, below which, low-level co-ancestry was not always reliably detected by BT SNPs. The Snipper analysis portal provides a comprehensive population dataset for the BT ancestry panel SNPs, comprising a 520-sample standardised reference dataset; 3445 additional samples from 1000 Genomes, HGDP-CEPH, Simons Foundation and Estonian Biocentre genome diversity projects; and 167 samples of six populations from in-house genotyping of individuals from Middle East, North and East African regions complementing those of the sampling regimes of the other diversity projects.

Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 °C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 ± 5% DC-RCY with a RCP > 99%, and was stable in injection buffer and human serum up to 3 h (>99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 ± 0.67% IA/g in the positive tumors compared with 0.42 ± 0.04% IA/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.

During the preparation of [68Ga]Ga-NOTA-sdAb at high activity, degradation of the tracers was observed, impacting the radiochemical purity (RCP). Increasing starting activities in radiolabelings is often paired with increased degradation of the tracer due to the formation of free radical species, a process known as radiolysis. Radical scavengers and antioxidants can act as radioprotectant due to their fast interaction with formed radicals and can therefore reduce the degree of radiolysis. This study aims to optimize a formulation to prevent radiolysis during the labeling of NOTA derivatized single domain antibody (sdAbs) with 68Ga. Gentisic acid, ascorbic acid, ethanol and polyvinylpyrrolidone were tested individually or in combination to find an optimal mix able to prevent radiolysis without adversely influencing the radiochemical purity (RCP) or the functionality of the tracer. RCP and degree of radiolysis were assessed via thin layer chromatography and size exclusion chromatography for up to three hours after radiolabeling. Individually, the radioprotectants showed insufficient efficacy in reducing radiolysis when using high activities of 68Ga, while being limited in amount due to negative impact on radiolabeling of the tracer. A combination of 20% ethanol (VEtOH/VBuffer%) and 5 mg ascorbic acid proved successful in preventing radiolysis during labeling with starting activities up to 1–1.2 GBq of 68Ga, and is able to keep the tracer stable for up to at least 3 h after labeling at room temperature. The prevention of radiolysis by the combination of ethanol and ascorbic acid potentially allows radiolabeling compatibility of NOTA-sdAbs with all currently available 68Ge/68Ga generators. Additionally, a design is proposed to allow the incorporation of the radioprotectant in an ongoing diagnostic kit development for 68Ga labeling of NOTA-sdAbs.

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [68Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining 68Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.

The study of DNA to predict externally visible characteristics (EVCs) and the biogeographical ancestry (BGA) from unknown samples is gaining relevance in forensic genetics. Technical developments in Massively Parallel Sequencing (MPS) enable the simultaneous analysis of hundreds of DNA markers, which improves successful Forensic DNA Phenotyping (FDP). The EU-funded VISAGE (VISible Attributes through GEnomics) Consortium has developed various targeted MPS-based lab tools to apply FDP in routine forensic analyses. Here, we present an evaluation of the VISAGE Basic tool for appearance and ancestry prediction based on PowerSeq chemistry (Promega) on a MiSeq FGx System (Illumina). The panel consists of 153 single nucleotide polymorphisms (SNPs) that provide information about EVCs (41 SNPs for eye, hair and skin color from HIrisPlex-S) and continental BGA (115 SNPs; three overlap with the EVCs SNP set). The assay was evaluated for sensitivity, repeatability and genotyping concordance, as well as its performance with casework-type samples. This targeted MPS assay provided complete genotypes at all 153 SNPs down to 125 pg of input DNA and 99.67% correct genotypes at 50 pg. It was robust in terms of repeatability and concordance and provided useful results with casework-type samples. The results suggest that this MPS assay is a useful tool for basic appearance and ancestry prediction in forensic genetics for users interested in applying PowerSeq chemistry and MiSeq for this purpose.

Since atherosclerotic plaques are small and sparse, their non-invasive detection via PET imaging requires both highly specific radiotracers as well as imaging systems with high sensitivity and resolution. This study aimed to assess the targeting and biodistribution of a novel fluorine-18 anti-VCAM-1 Nanobody (Nb), and to investigate whether sub-millimetre resolution PET imaging could improve detectability of plaques in mice. The anti-VCAM-1 Nb functionalised with the novel restrained complexing agent (RESCA) chelator was labelled with [18F]AlF with a high radiochemical yield (>75%) and radiochemical purity (>99%). Subsequently, [18F]AlF(RESCA)-cAbVCAM1-5 was injected in ApoE−/− mice, or co-injected with excess of unlabelled Nb (control group). Mice were imaged sequentially using a cross-over design on two different commercially available PET/CT systems and finally sacrificed for ex vivo analysis. Both the PET/CT images and ex vivo data showed specific uptake of [18F]AlF(RESCA)-cAbVCAM1-5 in atherosclerotic lesions. Non-specific bone uptake was also noticeable, most probably due to in vivo defluorination. Image analysis yielded higher target-to-heart and target-to-brain ratios with the β-CUBE (MOLECUBES) PET scanner, demonstrating that preclinical detection of atherosclerotic lesions could be improved using the latest PET technology.

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a “skull and crossbones” orientation. The lid of the 19th century coffin had brass tacks that spelled “JB55”, the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be “killed” by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname “Barber”. A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of “NB13,” found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.

The PD-1:PD-L1 immune checkpoint axis is central in the escape of cancer cells from anticancer immune responses. Monoclonal antibodies (mAbs) specific for PD-L1 have been approved for treatment of various cancer types. Although PD-L1 blockade has proven its merit, there are still several aspects that require further attention to fully capitalize on its potential. One of these is the development of antigen-binding moieties that enable PD-L1 diagnosis and therapy. We generated human PD-L1 binding single domain antibodies (sdAbs) and selected sdAb K2, a sdAb with a high affinity for PD-L1, as a lead compound. SPECT/CT imaging in mice following intravenous injection of Technetium-99m (99mTc)-labeled sdAb K2 revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and relatively low kidney retention, which is a unique property for radiolabeled sdAbs. We further showed using surface plasmon resonance that sdAb K2 binds to the same epitope on PD-L1 as the mAb avelumab, and antagonizes PD-1:PD-L1 interactions. Different human cell-based assays corroborated the PD-1:PD-L1 blocking activity, showing enhanced T-cell receptor signaling and tumor cell killing when PD-1POS T cells interacted with PD-L1POS tumor cells. Taken together, we present sdAb K2, which specifically binds to human PD-L1, as a new diagnostic and therapeutic agent in cancer management.

Genetic diversity of present American populations results from very complex demographic events involving different types and degrees of admixture. Through the analysis of lineage markers such as mtDNA and Y chromosome it is possible to recover the original Native American haplotypes, which remained identical since the admixture events due to the absence of recombination. However, the decrease in the effective population sizes and the consequent genetic drift effects suffered by these populations during the European colonization resulted in the loss or under-representation of a substantial fraction of the Native American lineages. In this study, we aim to clarify how the diversity and distribution of uniparental lineages vary with the different demographic characteristics (size, degree of isolation) and the different levels of admixture of extant Native groups in Colombia. We present new data resulting from the analyses of mtDNA whole control region, Y chromosome SNP haplogroups and STR haplotypes, and autosomal ancestry informative insertion-deletion polymorphisms in Colombian individuals from different ethnic and linguistic groups. The results demonstrate that populations presenting a high proportion of non-Native American ancestry have preserved nevertheless a substantial diversity of Native American lineages, for both mtDNA and Y chromosome. We suggest that, by maintaining the effective population sizes high, admixture allowed for a decrease in the effects of genetic drift due to Native population size reduction and thus resulting in an effective preservation of the Native American non-recombining lineages.

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories.

The translator incorporates the process of translation and, consequently, the mediation between languages and systems of culture. This mediation implies the interface of distinct dimensions such as the communicative or the semiotic. The translator is, at the same time, a reader of the source text and the producer of the target text. Bearing this in mind, this paper’s intention is to analyse the short-story “Love, Love, Love Alone”, by V. S. Naipaul, as the characters’ speech is mainly non-standard Caribbean English. Socio-cultural restricted elements such as this are a problem to translation due to expectancy norms in the target culture.

Neste artigo dedicamos a nossa atenção à temática dos resultados por acção, a qual tem merecido atenção especial por parte de diversas entidades normalizadoras, dada a sua apetência para aferir o progresso e o desempenho de uma entidade. A lnternational Accounting Standard (IAS) n.º 33, Earnings per Share e o Statement of Financial Accounting Standards (SFAS) n.º 128, com a mesma designação, são o resultado de 3 anos de esforços e de cooperação entre o International Accounting Standards Committee (IASC) e o Financial Accounting Standards Board (FASB), com o objectivo de clarificar o cálculo do resultado por acção, enunciando princípios para a sua determinação e apresentação que permitam melhorar as comparações à escala global. Depois de devidamente clarificados os objectivos, alcance e âmbito de aplicação dos resultados por acção, a nossa análise centra-se na determinação deste indicador, com especial destaque para os resultados (numerador), os quais são abordados à luz dos conceitos constantes da Directriz Contabilística (DC) n.º 20 - Demonstração dos Resultados por Funções. Deixaremos o estudo do denominador da fórmula de cálculo do resultado por acção para posterior desenvolvimento.

Neste artigo dedicamos a nossa atenção à problemática das técnicas de custeio, gestão empresarial e orçamentação baseadas na actividade num contexto em que as empresas, para alcançarem a excelência competitiva, se vêem na necessidade de melhorar continuamente o valor que oferecem aos clientes, identificando e eliminando as actividades que não acrescentam valor ao produto. É esta reflexão sobre as actividades das empresas um dos contributos das técnicas baseadas na actividade para a estratégia global das empresas. Depois de analisadas as características essenciais das técnicas baseadas na actividade, centramos a nossa atenção no desenvolvimento operacional das principais técnicas baseadas na actividade, designadamente: custeio, gestão empresarial e orçamentação, questionando-se o “real” valor das referidas técnicas. O objectivo é demonstrar que quaisquer sistemas contabilísticos e técnicas de gestão são válidos, desde que permitam à empresa acompanhar a evolução das tecnologias, o ciclo de vida dos produtos, o controlo de gestão, em suma, as mudanças das estratégias da empresa.

Neste artigo dedicamos a nossa atenção à problemática da harmonização contabilística internacional num contexto de crescente interpenetração e crescimento dos mercados económicos e financeiros, salientando-se as vantagens decorrentes da adopção de normas de contabilidade aceites internacionalmente e os obstáculos colocados pela diversidade de sistemas contabilísticos existentes. A harmonização contabilística internacional desenvolveu-se em duas vertentes, de acordo com a natureza do organismo que a leva a cabo: pública, isto é, sustentada no direito internacional público e privada ou profissional, emitida por instituições de peritos em contabilidade, sem instrumentos jurídicos que garantam a sua aplicação. Como exemplo da harmonização regional de carácter público mencionam-se as realizações da União Europeia, na sua tarefa de homogeneizar as legislações dos diferentes Estados Membros. Na harmonização de âmbito mundial e de carácter profissional destaca-se o trabalho desenvolvido pelo International Accounting Standards Committee (IASC), passando em análise as diferentes posturas face às suas normas, com especial referência para a adoptada pela União. Tudo isto com o objectivo específico de manifestar a importância que este organismo adquiriu nos últimos anos, como normalizador mundial da informação financeira.